The present invention relates to an anti-tumor agent comprising a disintegrin derived from snake venom, more specifically, to an anti-tumor agent comprising Salmosin which is a novel disintegrin containing Arg-Gly-Asp (RGD) sequence and derived from venom of Korean snake, Agkistrodon halys brevicaudus, as an active ingredient.
Tumor invasion and metastasis are the biological phenomena in which cancer cells lethally spread throughout the body. First, cancer cells detached from the primary site (e.g., epithelial tissue) breach the basement membrane separating them from other tissue layers. Some of these invasive cells can penetrate the basement membrane surrounding a blood vessel as well as the layer of endothelial cells lining it, which are then free to circulate via the bloodstream. Eventually, the cancer cells reach to a capillary, and adhere to and penetrate the capillary wall again, they can create a secondary tumor. Perhaps, fewer than one in 10,000 cancer cells that escape the primary site survives to form colony in another tissue (see: Erkki Ruoslahti, Scientific American, 72-77, September, 1996).
Therefore, tumor metastasis and invasion require adhesive interaction between cells and extracellular matrix (xe2x80x9cECMxe2x80x9d). In the course of tumor metastasis, tumor cells can cause endothelial cells to retract, exposing the subendothelial basement membrane and allowing the tumor cells efficiently to adhere to ECM proteins of the surrounding stroma (see: Hynes, R. O., Cell, 48:549, 1987). These matrix proteins promote cell adhesion by binding to specific cell surface receptors, including a member of integrin family.
In terms of structure, each integrin is a heterodimer consisting of xcex1 and xcex2 subunits which are noncovalently associated with each other. The xcex21 subfamily has been considered as a primary mediator of extracellular matrix adhesions. It has been reported that xcex21 integrins may have other functions, such as to mediate cellxe2x80x94cell adhesion directly (see: Larjava, H., et al., J. Cell. Biol., 110:803-815, 1990). The xcex22 subfamily that is found on leukocytes contains receptors mediating cellxe2x80x94cell interactions. The xcex23 subfamily includes the platelet glycoprotein IIb/IIIa complex and the vitronectin receptor, which may play an important role in the development of tumor invasiveness and malignancy (see: Albelda, S. M., et al., Cancer Res., 50:6757-6764, 1990).
The integrin receptor complex that is spanned the plasma membrane links the integral cytoskeletal network of a cell with the extracellular environment. Common or characteristic core sequences in cell adhesion molecules such as fibrinogen, vitronectin and laminin have been considered to contribute to cell adhesion and to the spread or integration of cells.
On the other hand, it was suggested that tumorigenesis and metastasis are closely associated with the biological role of integrins (see: Giancotti, F. G. and Rouslahti, E., Cell, 60:849-859, 1990; Hynes, R. O., Cell, 69:11-25, 1992; Nip, J., et al., J. Clin. Invest., 96:2096-2103, 1995).
Overexpression of fibronectin receptor xcex15xcex21 suppressed the transformed phenotype of Chinese hamster ovary cells. Integrin xcex15xcex21 was reduced in ras-transformed rodent cell (see: Plantefaben, L. C. and Hynes, R. O., Cell, 56:281-290, 1989) and superfibronectin that is a polymeric fibrillar form of fibronectin prevented tumor metastasis and tumor formation (see: Pasqualini, R., et al., Nature Medicine, 2:1197-1203, 1996).
Integrin xcex1vxcex23 is a specific marker of the most malignant cells, suggesting a crucial role of this adhesion receptor in the malignant growth of human melanoma (see: Albelda, S. M., et al., Cancer Res., 50:6757-6764, 1990). Integrin xcex1vxcex23 gene expression and the resulting adhesive phenotype are directly involved in the proliferation of human melanoma in vivo (see: Felding-Habermann, J. Clin. Invest., 89:2018-2022, 1992).
Angiogenesis is a biological process of forming new blood vessels as outgrowths from preexisting blood vessels (see: Folkman, J. and D""Amore, P. A., Cell, 87:1153-1155, 1996). This process plays a key role in the progression of solid tumor as well as normal development, wound healing and inflammation, and its regulation is contributed by vascular cell adhesion molecules in smooth muscle and endothelial cell (see: Nguyen, M., et al., Nature, 365:267, 1993).
The switch of angiogenic phenotype of tumor may be caused by losing balance between positive and negative modulators involved in neovascularization. Recently, it was reported that two cytokine-dependent pathways of angiogenesis were shown to exist and were defined by distinct vascular cell integrins, xcex1vxcex23 and xcex1vxcex25 that become expressed on angiogenic vascular cells where they play a critical role in angiogenesis induced by basic fibroblast growth factor (xe2x80x9cbFGFxe2x80x9d), tumor necrosis factor-alpha (TNF-xcex1), vascular endothelial growth factor (VEGF), and fragments of human tumors (see: Friedlander, M., et al., Science, 270:1500-502, 1995). Activation of xcex1vxcex23 integrin stimulates survival signal that facilitates blood vessel growth and differentiation indicating that signaling events by both cytokine and integrin receptors are closely associated with the growth of new blood vessels (see: Brooks, P. C., et al., Cell, 79:1157-1164, 1994).
On the other hand, several endogenous angiogenic inhibitors have been identified as followings: interferon-xcex1, -xcex3 (see: Friesel, R., et al., J. Cell. Biol., 104:689-696, 1987; Ezekowitz, R. A., et al., N. Engl. J. Med., 324:1456-1463, 1992); interferon-inducible protein 10(see: Angiolillo, A. L., et al., J. Exp. Med., 182:155-162, 1995; Strieter, R. M., et al., Biochem. Biophys. Res. Comm., 210:51-57, 1995); angiostatin and endostatin that specifically suppress endothelial cell proliferation (see: O""Reilly, M. S., et al., Cell, 79:315-328, 1994; O""Reilly, M. S., et al., Cell, 88:277-285, 1997); gro-xcex2 (see: Cao, Y., et al., J. Exp. Med., 182:2069-2077, 1995); the 16 kDa N-terminal fragment of prolactin (see: Clapp, C., et al., Endocrinology, 133:1292-1299, 1993); and, platelet factor-4 (see: Maione, T., et al., Science, 247:77-79, 1990; Gupta, S. K., et al., Proc. Natl. Acad. Sci., USA, 92:7799-7803, 1995).
It has been well known that disintegrins are a family of small proteins mainly derived from snake venoms (see: Niewiarowski, S., et al., Semin. Hematol., 31:289-300, 1994). Most of the disintegrins contain Arg-Gly-Asp (RGD) or Lys-Gly-Asp (KGD) sequence which is the structural motif recognized by a platelet fibrinogen receptor xcex12bxcex23, and also act as a potent antagonist of several integrins including xcex1vxcex23 and xcex15xcex21. There are several reports demonstrating that disintegrins containing the RGD sequence inhibit tumor metastasis by blocking tumor cell adhesion to ECM (see: Trikha, M. et al., Cancer Res., 54(8):4993-4998, 1994).
Integrin xcex1vxcex23 was identified as a marker of angiogenic blood vessels in chick embryo and human (see: Brooks, P. C., et al., Science, 264:569-571, 1994). Monoclonal antibody against xcex1vxcex23 was able to perturb angiogenesis by inducing apoptosis in the endothelial cells of the newly formed blood vessels. Application of synthetic peptides containing the RGD sequence that inhibit ligand binding to integrin xcex1vxcex23 suppressed tumor-induced angiogenesis on chick chorioallantoic membrane (xe2x80x9cCAMxe2x80x9d) (see: Brooks, P. C., et al., Cell, 99:1157-1164, 1994), and also suppressed the function of angiogenin which assists adhesion and diffusion of endothelial cells. Recently, triflavin, a disintegrin derived from snake venom, is reported to inhibit angiogenesis induced by TNF-xcex1. These findings suggest that disintegrins, synthetic RGD peptides and anti-xcex1vxcex23 monoclonal antibody may be developed as a potent anti-tumor agent.
In line with the previous reports, the present inventors isolated Salmosin derived from venom of Korean snake, Agkistrodon halys brevicaudus, and characterized that: it is a novel protein of 7.5 kDa having a strong inhibitory activity against platelet agglutination (see: Korean Patent No. 142606, SEQ ID NO:1); and, it contains RGD sequence which is known to inhibit ligand binding to integrin xcex1vxcex23.
Under the circumstances, there are strong reasons for exploring and developing an anti-tumor agent comprising an active ingredient of Salmosin derived from venom of Agkistrodon halys brevicaudus. 
The present inventors isolated and purified Salmosin from venom of Korean snake Agkistrodon halys brevicaudus, cloned cDNA thereof and overexpressed recombinant Salmosin in E. coli. Then, they have developed an anti-tumor agent comprising an active ingredient of Salmosin which is a disintegrin containing RGD sequence, based on the finding that Salmosin can strongly inhibit tumor angiogenesis which is essential to cancer cell growth and metastasis without affecting proliferation of normal endothelial cell.
A primary object of the present invention is, therefore, to provide an anti-tumor agent comprising an active ingredient of Salmosin derived from venom of Korean snake, Agkistrodon halys brevicaudus.